Genome-wide transcription-coupled repair in Escherichia coli is mediated by the Mfd translocase

Abstract

We used high-throughput sequencing of short, cyclobutane pyrimidine dimer-containing ssDNA oligos generated during repair of UV-induced damage to study that process at both mechanistic and systemic levels in Escherichia coli Numerous important insights on DNA repair were obtained, bringing clarity to the respective roles of UvrD helicase and Mfd translocase in repair of UV-induced damage. Mechanistically, experiments showed that the predominant role of UvrD in vivo is to unwind the excised 13-mer from dsDNA and that mutation of uvrD results in remarkable protection of that oligo from exonuclease activity as it remains hybridized to the dsDNA. Genome-wide analysis of the transcribed strand/nontranscribed strand (TS/NTS) repair ratio demonstrated that deletion of mfd globally shifts the distribution of TS/NTS ratios downward by a factor of about 2 on average for the most highly transcribed genes. Even for the least transcribed genes, Mfd played a role in preferential repair of the transcribed strand. On the other hand, mutation of uvrD, if anything, slightly pushed the distribution of TS/NTS ratios to higher ratios. These results indicate that Mfd is the transcription repair-coupling factor whereas UvrD plays a role in excision repair by aiding the catalytic turnover of excision repair proteins.

Keywords: Mfd; UvrD; XR-seq; mutagenesis; transcription-coupled repair.

Fig. 1.

Scheme for analyzing repair of the E. coli genome. Cells are irradiated with UVC (254 nm) and then incubated for a period to allow nucleotide excision repair. Cells are then harvested and cooled on ice, and then the Hirt procedure (23) is used to separate relatively small DNA molecules from genomic DNA and cellular debris. From the preparation of small DNA molecules, DNA molecules containing cyclobutane pyrimidine dimers (CPDs) are then isolated by immunoprecipitation with an anti-CPD–specific antibody. The isolated products may be analyzed in two ways. To detect the overall excision of damage from the genome, samples may be directly end-labeled with ³²P and subjected to denaturing polyacrylamide gel electrophoresis. To map the sites of DNA excision repair throughout the genome at nucleotide resolution, products may be analyzed by high-throughput sequencing after they are ligated to adaptors, immunoprecipitated again with anti-CPD antibody, photoreactivated, amplified by PCR, and gel-purified (25).

Fig. 2.

Analysis of nucleotide excision repair products from E. coli by excision assay. Cells were irradiated with 100 J/m² (A and B) or 120 J/m² (C) and then incubated for the indicated times (in A) or for 5 min (B and C). Repair products containing a CPD were isolated from cells, end-labeled with ³²P, and resolved by denaturing polyacrylamide gel electrophoresis. The gel images show a predominantly 13-mer size excision product obtained from WT cells, which is consistent with results from in vitro analysis (27). This 13-mer size excision product is simultaneously generated and degraded in vivo, and a reduction in the amount of the 13-mer product seen after 30 min is consistent with an ∼30-min time course to complete nucleotide excision repair in E. coli cells (3). Strain STL4150 lacks the major ssDNA exonucleases, and the images in A and B show that, in STL4150 cells, there is limited degradation of the 13-mer past the 10-mer stage. In uvrD mutant cells (B and C), there is an elevated level of 13-mer product. The UvrD protein is the major helicase in E. coli. In nucleotide excision repair, UvrD catalyzes the displacement of the damage-containing 13-mer excision product and initiates displacement of the UvrB and UvrC proteins from the genome. Consequently, in these cells, the 13-mer remains annealed to the genome where it is resistant to nucleases. This accumulation of the 13-mer is observed even though uvrD mutant cells excise the 13-mer slowly because turnover of UvrB and UvrC is slow. The 12-mer marker DNA shown in the images contains a CPD and was end-labeled with polynucleotide kinase. The 50-mer (1 fmol) was included in each sample before end-labeling, as an internal control.

Fig. 3.

Length distribution and nucleotide composition of the reads. (A) Length distribution of the isolated excised fragments for four strains. The lengths of the principal excised products are 10, 12, or 13 nt. The remaining reads are considered as background. (B and C) Position-specific nucleotide frequencies of the 13-mer (B) and 10-mer (C) reads from parental strain STL4150. Results are from the first of two experiments.

Fig. 4.

Frequency distribution of Log2-transformed TS/NTS repair in all annotated genes. (A) E. coli genes in four different strains colored by sense strand transcription levels going from lowest quartile RNA-seq count colored in red, to orange, to green, to the highest transcription quartile, in blue. Theoretically, the more weakly transcribed genes are more likely to exhibit repair profiles strongly influenced by antisense transcription and by repair hotspots. The means for each sample are 1.16 (Parental), 1.14 (phr⁻), 0.68 (mfd⁻), and 1.17 (uvrD⁻), and all of them are different from 0 based on one sample t test with P values <0.01. In Exp. 2, means for each sample were 1.09, 1.14, 0.68, and 1.04. (B) Top 25% most transcribed genes are plotted. Means for parental, phr, mfd, and uvrD cells are 1.41, 1.46, 0.53, and 1.61 for Exp. 1 (plotted) and 1.41, 1.46, 0.54, and 1.21 in Exp. 2, respectively. The vertical black line represents the border where TS repair level is equal to NTS repair.

Fig. 5.

Scatter plots of TS/NTS repair signal in all annotated genes. (A) Log2-transformed TS/NTS repair ratio, which represents the TCR signal, is plotted for phr⁻, mfd⁻, and uvrD⁻ strains on the y axis vs. the parental strain (x axis). The dashed red line is the line of equality (x = y) representing equal TCR signal in the two strains. In the phr⁻ strain, although most genes exhibit mildly elevated TS/NTS compared with parental, the difference is not significant. In the mfd⁻ strain, most genes have a lower value of TS/NTS repair compared with WT because blocked RNAP inhibits TS repair. The parental TS/NTS mean value is 1.71-fold higher than mfd⁻ (P < 0.01). Note that the loss of Mfd is associated with more NTS repair (relative to parental cells) even in genes that, in the parental cell line, exhibit more NTS repair than TS repair (left half of plot). In uvrD⁻ strains, the mean of TS/NTS is mildly but significantly higher than in WT (P = 8e−08) in this experiment, but note that there was no difference in Exp. 2. (B) The correlations between TS repair (fragments per 100 TT sites per million reads, on the y axis) and sense strand transcript levels (fragments per 1 kb per million reads, on the x axis). The correlations in WT, phr, and uvrD cells are significant, with correlation coefficients 0.36, 0.36, and 0.22, respectively (P values < 2e−16). In mfd, a mild negative correlation was observed (rho = −0.07, P = 2e−5). (C) The ratios of mutant TS/NTS repair over WT TS/NTS repair as a function of transcription level. The values on both axes are log2-transformed. Transcription levels are RNA-seq read counts that are compiled from the raw data produced by Thomason et al. (36) (SRR1173967). As opposed to other strains, in the mfd⁻ strain, there is a strong correlation (P < 2e−16) with the Spearman’s rho of −0.55. The blue trend lines (B and C) were drawn based on simple linear regression models.

Fig. 6.

XR-seq and RNA-seq patterns of exemplified operons and genes. In all panels, the first four green-colored rows represent the XR-seq reads of the four strains studied aligned with the minus strand of the reference genome (E. coli K-12 MG1655 genome with NCBI accession NC_000913.2). The fifth green bar shows the RNA-seq reads aligned with the plus strand. Blue bars represent the opposite strand. For the genes that are on the plus strand (A, C, and D), the green XR-seq reads correspond to the transcribed strand repair where green RNA-seq reads represent the RNA products that are due to the sense transcription. Blue XR-seq data illustrate repair of the coding (plus) strand, and blue RNA-seq reads represent the antisense transcription. The opposite is true for the genes on the minus strand (B). The y axis is scaled to show 105 counts for each bar except for the right panel of D, where the y axis is scaled up to 2,000. (A) The illustrated 6-kb genomic window is between 4,164 and 4,170 kb, which is an rRNA operon rrnB with 16S (rrsB), 23S (rrlB), and 5S (rrfB) rRNA genes, as well as the glutamate tRNA (gltT) gene. This operon was selected to represent a high level of transcription so that the Mfd effect is drastically visible. (B) Chemotaxis operon (1964.25 to 1973.5 kb) with moderate level of transcription. (C) The rpoB gene (4179.2 to 4183.35 kb) displays an amplified repair signal in the uvrD strain. (D) The insL1 gene (2512.3 to 2513.5 kb) with antisense transcription in the 3′ end (Left).

Fig. S1.

Reproducibility of the mutation effect on TCR (A) phr^–, (B) mfd^–, and (C) uvrD^–. The x and y axes represent Exp. 1 and Exp. 2, respectively. Axis values are the log2-transformed ratio of mutant/Parental TS/NTS values. Pearson correlation coefficients are 0.43 (phr^–), 0.85 (mfd^–), and 0.81 (uvrD^–). Blue bars along the axes represent density distribution of data points.

Fig. 7.

Schematic representation of the model for the nucleotide excision repair in E. coli controlled by Mfd and Uvr proteins in the dark. When there is an alternative path, the arrow widths indicate the pathway preference: The thicker arrows are preferred over the thinner ones. The green-shaded pathway indicates TCR, and, in the absence of Mfd, this pathway is blocked. The yellow-shaded arrow indicates the enhanced recruitment of UvrA₂B₁ with the aid of Phr, which is more prevalent with lesions in the NTS because TS lesions undergo TCR. The purple-shaded pathway is blocked in the absence of UvrD, and UvrBC complex doesn’t turn over.